Analysis of fatty acids in mouse cells using reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography (RP-HPLC) method wa s developed to analyze various fatty acids in recombinant mouse L cells. Th ese fatty acids were the metabolites of oleic acid. A process was developed to extract fatty acids from the cell samples before RP-HPLC analysis. The samples were first saponified with 0.5 M NaOH in 96% ethanol then extracted with acidified ethyl acetate. After extraction, the sample was dried and d issolved in HPLC-grade methanol. After centrifugation to remove insoluble i mpurities, the sample was applied to a C-18 RP-HPLC column using a gradient of acetonitrile (ACN)-H2O. The eluted fatty acids were monitored by ultrav iolet (UV) absorption at 195 nm and identified by retention time and adsorp tion spectrum comparison. This method successfully resolved various fatty a cids and provided a tool for the elucidation of the fatty acid metabolic pa thway in the cells.